Project Summary/Abstract In the Kleiner laboratory we study metabolism, physiology and interactions in microbial symbioses and host- associated microbiota. For this we combine a diversity of cultivation independent approaches ? such as metagenomics, metabolomics, metaproteomics and single cell imaging ? with cultivation-based approaches including heterologous gene expression, biochemical assays and other in vitro measurements. Our research has a strong focus on the development of high-resolution mass spectrometry driven metaproteomics for the large-scale identification and quantification of proteins in host-associated microbiota. During the next five years, I plan to continue using these approaches to study intestinal microbiota responses to, and mechanisms of interaction with, external substrates (diet) and host-derived substrates (host compound foraging). I hypothesize that different dietary protein sources and host-derived compounds will have vastly different impacts on the microbiota and thus need to be considered when studying the interconnection of diet, the microbiota, and host health. I propose to use metaproteomics, complemented with metagenomics and metabolomics, to (1) identify and quantify the substrates that are used and converted by microbiota members and (2) determine how these substrates impact community composition and functional interactions with other microbiota members and the host. This research will provide urgently needed insight into the functional impacts of substrates consumed by gut microbiota by optimizing and deploying novel approaches for the reproducible, large-scale characterization of host, diet and microbial proteins in the intestinal tract. My long-term goals are to develop metaproteomic approaches that allow us to quantitatively and reproducibly determine functional interactions in microbial communities, and to define critical interactions between the microbiota and dietary proteins that will inform the development of therapeutic interventions. These approaches will also be powerful tools for studying any disease associated with the human microbiome in and on different body sites and microbial communities that humans interact with in their environment and that potentially impact health.